Methods of determining the sequence of nucleotides in nucleic acids have undergone substantial changes from the original adoption of gel-based Sanger sequencing, through four color capillary electrophoresis based approaches, both of which relied upon the electrophoretic separation of nested synthesis fragment sets to identify sequentially terminated synthesis products, and as a result, identify each successive base in the sequence. Newer approaches to sequencing rely upon “sequencing by incorporation” where each base is identified sequentially, as it is added in a primer extension reaction. These range from pyrosequencing and other related methods that add a single base at each step and look to see if it was incorporated, to processes that add multiple different types of nucleotides each labeled with a different fluorescent dye, and identify which base was incorporated based upon the dye incorporated at any given step. Typically, such processes require an iterative or step by step process that employs nucleotides that include extension terminating groups, such that after a single incorporation event, no new bases are added until the added base can be identified. The terminating group is then removed and the next extension step is allowed to proceed.
In still more elegant methods, individual molecular complexes are observed in real time, as they incorporate labeled nucleotides. The incorporation event provides a characteristic optical signal that, along with a spectrally distinct dye, identifies both the incorporation event and the type of base incorporated. In such methods, the labeling group is often provided coupled to the phosphate chain of the nucleotide analog beyond the alpha phosphate, resulting in cleavage of the label from the nucleotide upon incorporation. This allows both the synthesis of an entirely native strand of nucleic acid, and the release of a labeling group that might otherwise confound the observation and analysis.
The present invention provides improved compositions, methods and systems for performing single molecule real time analyses, and particularly single molecule, real time nucleic acid sequence analysis.